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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: Clear colourless liquid
- Analytical purity: 97.8%
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Storage condition of test material: at room temperature in the dark
- Other: specific gravity: > 0.94 g/cm3
Analytical monitoring:
yes
Details on sampling:
Concentrations:
- Range-finding test: 10 mg/L
- Final test: at the start of testing (control, 2.2, 22, and 100 mg/L) and at the end of testing (all test concentrations)
Sampling: duplicate 10-mL samples from each sampled concentration
Sampling frequency:
- Range-finding test: t=0, t=24, t=72
- Final test: see above
Sampling storage: not applicable, immediate analysis
Vehicle:
no
Details on test solutions:
- Preparation of test solutions started with a stock solution of 100 mg/L in test medium, applying a 15-minute stirring period.
- Preparation of lower test concentrations through subsequent dilutions of the stock in test medium.
- Since pH of stock solution was quite high (alkaline) (outside optimum range for testing with algae (6.0-9.0)), pH was adjusted to 8.1 with 1 N HCl in the final test to exclude possible pH related effects.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): 3-4 days (from pre-culture)
- Method of cultivation: stock cultures started by inoculating M1 growth medium with algal cells from a pure culture on agar; suspensions continuously aerated, kept at 23±2°C, and continuous illumination (60-120 µE2/m2/s, wavelength 400-700 nm)

ACCLIMATION
- Pre-culture in M2 (ISO) medium during 3 to 4 days prior to testing
- Pre-culture medium is same as test medium
- Pre-culture kept at same conditions as test conditions
- Initial density of pre-culture: 2E04 cells/mL.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
23.5-24°C (measured in temperature control vessel)
pH:
8.0-10.0 (measured at the beginning and at the end of the test).
pH of the control, 2.2 and 10 mg/L test groups was > 9.0 and in addition pH of the 2.2 mg/L test group increased > 1.5 unit during the test period. Since this was related to a relatively high rate of algal growth this deviation was not considered to have affected the validity of the test.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Range-finding test:
- Nominal concentrations: Control + 0.1-1.0-10-100 mg/L
- Measured concentrations (only 10 mg/L): 71-104% of nominal
Final test:
- Nominal concentrations: Control + 2.2-4.6-10-22-46-100 mg/L
- Measured concentrations at start of testing: 96-88% of nominal for 22 and 100 mg/L and 44% of nominal for 2.2 mg/L
- Measured concentrations at end of testing: 98-107% of nominal for 4.6 up to 100 mg/L and 47-51% of nominal for 2.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test medium
- Aeration: no, but continuous shaking
- Static system
- Initial cells density: 1E04 cells/mL
- Control end cells density: in the controls, cell density increased by an average factor of > 16 within 3 days (106.91-143.68 x 10^4 cells/mL)
- Replicates: 3 replicates of each test concentration; 6 replicates of the blank-control; 1 replicate of the highest concentration without algae

GROWTH MEDIUM
- Standard medium used in test and pre-culture: yes, M2 (ISO)
- Standard medium used for culturing: yes, M1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q (tap water purified by reverse osmosis (milli-RO) and passed over activated carbon and ion-exchange cartridges)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH of test media adjusted to 8.1 with 1 N HCl prior to testing, no adjustment during the test
- Photoperiod: continuous illumination
- Light intensity and quality: 80 to 115 µE/m2/s (TLD-lamps of the type Cool-white of 30 Watt)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Cell density was determined at 24, 48, and 72 h
- At the beginning of the test, cells were counted by microscope, using a counter chamber.
- Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength = 20 mm), Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank.
- Quantification of cell densities was based on a calibration curve.

TEST CONCENTRATIONS
- Range-finding test: Control + 0.1-1.0-10-100 mg/L
- Final test: Control + 2.2-4.6-10-22-46-100 mg/L
- Results used to determine the conditions for the definitive study: the range-finding study showed that the expected EC50 for cell growth inhibition was between nominal concentrations of 10 and 100 mg/L and for growth rate reduction was > 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI = 7.3-52 mg/L
Details on results:
Growth rate:
72-h NOEC, LOEC, EC10 and EC50 = 4.6, 10, 20, and > 100 mg/L, respectively.

Biomass:
72-h NOEC, LOEC, EC10 and EC50 = 4.6, 10, 4.4, and 44 mg/L, respectively.
Results with reference substance (positive control):
72-h EbC50 = 0.59 mg/L (95% CI = 0.38-0.92 mg/L), i.e., within ISO range of 0.20 to 0.75 mg/L
72-h ErC50 = 0.89 mg/L (95% CI = 0.56-1.4 mg/L), i.e., within ISO range of 0.60-1.03 mg/L
Reported statistics and error estimates:
NOEC determined using ANOVA, Tukey test, TOXSTAT Release 3.5, 1996.
EC50 and EC10 calculated based on linear regression analysis of the percentages of growth rate reduction versus logarithms of corresponding nominal concentrations of test substance.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study with the unicellular freswhater green alga Selenastrum capricornutum (Pseudokirchneriella subcapitata), XTJ 568 reduced growth rate significantly at nominally 10 mg/L and higher. The 72-h NOEC, LOEC, EC10 and EC50 based on growth rate were 4.6, 10, 20 and > 100 mg/L, respectively. The test results are valid and the study can be considered a Klimisch 1 study.

Description of key information

The Klimisch 1 study by Bouwman (2004) performed at NOTOX is considered the key study. This algal growth inhibition test with the unicellular green alga Selenastrum capricornutum (Pseudokirchneriella subcapitata), performed according to ISO Guideline 8692, EU Method C.3 and OECD Guideline 201,  yielded a 72-h NOEC and EC50 (based on growth rate reduction) of 4.6 and > 100 mg/L, respectively. These values can be used as key values for PNEC derivation.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
4.6 mg/L

Additional information

A single algal growth inhibition study is available (Bouwman, 2004). Under the conditions of this study with the unicellular freshwater green alga Selenastrum capricornutum (Pseudokirchneriella subcapitata), the substance reduced algal growth rate as well as biomass significantly at nominally 10 mg/L and higher. The 72-h NOEC, LOEC, EC10 and EC50 based on algal biomass were 4.6, 10, 4.4 and 44 mg/L, respectively, whereas the 72-h NOEC, LOEC, EC10 and EC50 based on algal growth rate were 4.6, 10, 20 and > 100 mg/L, respectively. The results of this test are valid and the study is considered a Klimisch 1 study.